This proposal seeks to extend studies of Reverse transcriptase (RT) from synthetic substrates to more complex physiologically relevant substrates and systems including natural tRNA31ys and NCp7 protein. These studies will focus to increase the mechanistic understanding, at a molecular level, of the enzymatic activities of RT and the interaction with clinically significant nucleoside inhibitors, the mechanism of resistance to these compounds that develops through specific RT mutations, the mechanism of differential selectivity between the mechanistic exploration of a recently designed class of "bifunctional" inhibitors that span the nucleoside and non-nucleoside binding sites. It is the ultimate goal of these studies to develop an in-depth understanding of the enzyme mechanism of HIV1-RT and the interaction with inhibitors as well as to uncover the underlying mechanisms of drug resistance and toxicity that could ultimately lead to compounds that are less toxic, more selective, and hence more effective as therapeutics. The specific aims of the current proposal are: 1) Continue our mechanistic studies on the catalytic activities of HIV reverse transcriptase at a molecular level by providing a kinetic and thermodynamic understanding of the events involved in the process of tRNA3 Lys initiation. 2) Examine Mechanisms of Inhibition of HIV1-RT by clinically significant nucleoside analogs. 3) Explore the Mechanistic Basis for Similarity Between the Mitochondrial gamma DNAPolymerase and HIV Reverse Transcriptase. 4) Design and Evaluate Novel Bifunctional HIV-1 Inhibitors created by linking nucleoside and non-nucleoside analogs.